Iron rescues glucose-mediated photosynthesis repression during lipid accumulation in the green alga Chromochloris zofingiensis

Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (−Fe). Cells grown in −Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.

cofactor-containing THI players suggests C. zofingiensis displays an evolutionary novel iron sink priority in WT−Fe+Glc (see Fig. 9).
The example of unique highest-in-heterotrophy proteins show how iron priorities during deficiency may diverge according to an organism's environment and evolutionary programming.The iron-cofactor proteins for thiazole biosynthesis and PAO5 were uniquely upregulated in WT−Fe+Glc and could be iron sinks when several other iron-rich proteins are depleted in heterotrophy.In particular, the THI1/4 family has duplicated to three copies in C. zofingiensis, all of which are induced in heterotrophy (Fig. 9).This protein family is known to be metabolically costly due to catalyzing only a single reaction turnover per protein 3 .It is surprising that C. zofingiensis upregulates these biosynthetic proteins in −Fe+Glc despite their metabolic and iron costs, especially if the THI1/4 genomic duplications evolutionarily increased this protein's concentration or led to neo-functionalization.Further research may unveil the importance of the thiazole moiety to heterotrophic metabolism, which could include novel enzymatic uses for thiazole-derived products in C. zofingiensis.

Proteomic Mass-Spectrometry Protein extractions and processing for proteomics
For protein extraction, each cell pellet was resuspended in 200 µl of H2O and transferred to 2 mL pre-filled Micro-Organism Lysing Mix glass bead tubes and disrupted in a Bead Ruptor Elite bead mill homogenizer (OMNI International, Kennesaw, GA) at speed 5.5 m/s for 45 s.After bead beating, the lysate was immediately placed on ice and then centrifuged at 1,000 x g for 10 mins at 4°C.To separate the proteins, metabolites and lipids, 1 mL cold (-20°C) 2:1 chloroform:methanol (v/v) was pipetted into a chloroform-compatible 2 mL Sorenson MulTI™ SafeSeal™ microcentrifuge tube (Sorenson Bioscience, Salt Lake City, UT) on ice.The 200 µl of sample homogenate was then added to the Sorenson tube and vigorously vortexed.The sample was placed on ice for 5 min and then vortexed for 10 s followed by centrifugation at 10,000 x g for 10 min at 4°C.The protein interface had 1 mL of cold 100% methanol added to each sample, was vortexed and centrifuged again at 10,000 x g for 10 min at 4°C to pellet the protein.The methanol was then decanted off, and the samples were placed open in a fume hood to dry for ~10 min.
The protein pellet was dissolved in 200 µl of 8 M urea and vortexed into solution.A bicinchoninic acid (BCA) assay (Thermo Scientific, Waltham, MA USA) was performed to determine protein concentration.Following the assay, 10 mM dithiothreitol (DTT) was added, and the samples were incubated at 60°C for 30 min with constant shaking at 800 rpm.Samples were then diluted 8-fold in preparation for trypsin digestion.100 mM NH4HCO3, 1 mM CaCl2 and sequencing-grade modified porcine trypsin (Promega, Madison, WI) were added to all protein samples at a 1:50 (w/w) trypsin-to-protein ratio for 3 h at 37˚C with constant shaking at 450 rpm.Digested samples were desalted using a 4-probe positive pressure Gilson GX-274 ASPEC™ system (Gilson Inc., Middleton, WI) with Discovery C18 100 mg/1 mL solid phase extraction tubes (Supelco, St. Louis, MO), using the following protocol: 3 mL of methanol was added for conditioning followed by 2 mL of 0.1% trifluoroacetic acid (TFA) in H2O.The samples were then loaded onto each column followed by 4 mL of 95:5: H2O: acetonitrile, 0.1% TFA.Samples were eluted with 1 mL 80:20 acetonitrile:H2O, 0.1% TFA.The samples were concentrated down to ~100µL using a SpeedVac, and a final BCA assay was performed to determine the peptide concentration.An equal mass of each sample was aliquoted into fresh centrifuge tubes, dried completely in a SpeedVac and then stored at -80°C until isobaric labeling.

TMT isobaric tag labeling
Each sample was diluted in 500 mM HEPES, pH 8.5 to a concentration of 5 µg/µl and labeled using amine-reactive Thermo Scientific Tandem Mass Tag (TMT10) Isobaric Mass Tagging Kits (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions.Briefly, 250 μL of anhydrous acetonitrile was added to each 5 mg reagent, vortexed and allowed to dissolve for 5 min with occasional vortexing.Reagents were then added to the samples and incubated for 1 h at RT with shaking at 400 rpm.Each sample was diluted to 2.5 µg/µl with 20% acetonitrile, and the reaction was quenched by adding 8 μL of 5% hydroxylamine to the sample with incubation for 15 min at RT with shaking at 400 rpm.Samples within each set were combined and completely dried in the SpeedVac.Each sample was cleaned using C18 50 mg/1 mL solid phase extraction tubes as described above and again assayed with BCA to determine the final peptide concentration.

High pH RP C-18 fractionation
TMT samples were diluted to a volume of 900 μL with 10 mM ammonium formate buffer (pH 10.0), and resolved on a XBridge C18, 250x4.6 mm, 5 μm with 4.6x20 mm guard column (Waters, Milford, MA).Separations were performed at 0.5 mL/min using an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA) with mobile phases (A) 10 mM ammonium formate, pH 10.0 and (B) 10 mM ammonium formate, pH 10.0/acetonitrile (10:90).The gradient was adjusted from 100% A to 95% A over the first 10 min, 95% A to 65% A over minutes 10 to 70, 65% A to 30% A over minutes 70 to 85, maintained at 30% A over minutes 85 to 95, reequilibrated with 100% A over minutes 95 to 105, and held at 100% A until minute 120.Fractions were collected every 1.25 minutes (96 fractions over the entire gradient).Every other row was concatenated into 24 fractions and dried completely, and 25 µL of 25 mM ammonium bicarbonate was added to each fraction for storage at -20°C until LC-MS/MS analysis.

Lipid extraction and thin layer chromatography.
The procedures for visualizing lipids by TLC was adapted from Supplementary Reference 4. Cell pellets from 2mL of culture were flash frozen.Cells were lysed in 2 mL screw-cap tubes with Lysing Matrix D beads through a MP Fastprep-24 TM 5G bead beater (settings 6.5 m/s, 60s, 2 rounds) with dry ice in the CoolPrep TM adapter.The frozen cell pellets were given 0.5 mL (except the high biomass WT+Fe+Glc sample, where extraction volumes were doubled) of 2:1 chloroform:methanol solution with 0.01% butylated hydroxytoluene and were vortexed for 5 min.133 µL (266 for WT+Fe+Glc) of 0.73% (w/v) NaCl was added to the cultures, which were centrifuged for 2min at 14,000 rpm (20317 RCF).The lower solvent with extracted lipids was transferred Eppendorf tubes.The volume of extracted lipids added to Supelco TLC Silica gel 60 F254 (1.05715.0001)pretreated with developing solution was adjusted so that the same culture biomass (2.51 x 10 7 µm 3 ) per extraction solution was loaded on plate.2.5 µL of lipid extracted olive oil (25 µL Good & Gather, extracted in 0.5 mL of 2:1 chloroform:methanol solution as above) was also loaded as a TAG standard.The plate was developed with hexane:diethyl ether:acetic acid (91:30:1.3)which shows strong separation of TAGs [8].The plate was lightly sprayed with 25% H2SO4 in 50% Ethanol with a Aldich R flask-type sprayer (75 mL), dried for 10 minutes, than charred in a ~100°C oven until the lipid bands were prominent.The TLC plate was photographed while being illuminated by Fotodyne UV Transilluminator.

Carotenoid and chlorophyll detection by high performance liquid chromatography
5 mL of culture at the 84 h sampling time point had growth measurements were spun down, the supernatant was removed, and remaining cell pellet was flash frozen.Samples were resuspended in 100% HPLC-grade acetone and homogenized with Matrix D beads in a FastPrep-24 5G™High-Speed Homogenizer (6.5 m s-1 for 2 x 60 s, MP Biomedical).The pigmented supernatant was collected after centrifugation (2 min, 1500 g, 4°C) and the acetone extraction was repeated until the cell pellet was white and supernatant was mostly clear towards the last extractions.All steps were held on ice, in the dark as much as possible.Sample runs through the HPLC were conducted by a previously described chlorophyll and carotenoid quantification protocol [4][5][6][7] on an Agilent 1100 HPLC system.Supplementary Fig. 5 Low abundance of HXK1 is detected in hxk1 strains.a. Relative protein abundance of HXK1 across strains (n = 3-4, individual data points shown).Glc and Fe had minimal impact on abundance at this 84 h timepoint.B-d.Unique Peptides Aligned to the HXK1 protein sequence.For each strain, each rectangle represents uniquely detected peptides and their aligned to their position on the protein sequence (x-axis).Y-axis position signifies relative abundance of each peptide and each amino acid is colored according to RasMol (2.7.5) color schemes.The black rectangle in the hxk1 strains signifies the site where the C471CG insertion is supposed to confer a frame shift (AA position 55) to the mutant protein's predicted early stop (AA position 65).Most peptides downstream align to downstream of the frame shift and stop.Plots b-d shows the mean unique peptide abundance within +Fe−Glc conditions per strain, but peptides aligned downstream of the frameshift mutation occur in all conditions.Source data are provided in the Source Data File.consumption in the dark) and the light (basis of net oxygen consumption).Change in oxygen is negligible in WT-Fe+Glc during dark to light transition.Individual lines represent individual culture oxygen measurements.Source data are provided in the Source Data File.Supplementary Fig. 9 Significance of ortholog overlap in photosynthetic regulation with reference organisms.a. Heatmap ortholog enrichment overlap analysis between the highly stringent and less stringent photosynthesis lists (y-axis) in C. zofingiensis and several photosynthesis associated lists developed in C. reinhardtii [8][9][10][11] (x-axis).The number of total distinct ortholog groups per photosynthesis gene list is in parenthesis on axes labels.The number of overlapping orthologs groups found in each species' gene list is written in heatmap block, which is colored by the p-value of overlap significance determined by the Fisher exact test.Only nucleus-encoded genes were included in analysis due to lack of C. reinhardtii plastid-encoded genes used in transcriptome and phylogenomic analysis.b.Significance heatmap of C. zofingiensis lowest in heterotrophy and their orthologs upregulated de-etiolation in the proteome measured in A. thaliana 12.The x-axis considers various stringency of induction, with log2 fold change (FC) greater than >0.5, >1, or >2 from the 0 h etiolated time point in both 24 and 96 h.Plastid encoded proteins are included in analysis.c. C. zofingiensis lowest in heterotrophy ortholog with Zea mays photosynthetic development upregulated along basipetal axis.M5, M9, and M15 refer to various photosynthetic tissues and their upregulation in expression compared to the undeveloped photosynthetic tissues below the ligule (M1) 13  C. zofingiensis rapidly upregulates (yellow scale) a complete dnFAS pathway in both photoautotrophic nitrogen deprivation 15 and glucose addition 6 , despite different impacts of these nutrients on overall biomass.Nitrogen deprivation in C. reinhardtii 16 leads to a concerted downregulation of this entire pathway (blue).Transcriptional data is log2 normalized the ratio of time-controlled +Glc sample over condition not treated with Glc.Nitrogen conditions in both species are normalized to a 0 h timepoint.Source data are provided in the Source Data File.
. d. C. zofingiensis lowest in heterotrophy orthologs with Oryza sativa photosynthetic development along basipetal axis.Like in Z. mays R3, R7, and R11 are photosynthetically developed tissue whose upregulation in expression is compared to the undeveloped photosynthetic tissues below the ligule (R1) 13 .Source data are provided in the Source Data File.Supplementary Fig. 10 Thiamine and methionine biosynthetic processes are upregulated in heterotrophy.a. Proteomics heatmap showing upregulation of THIC, all C. zofingiensis copies of THI4 (manually labeled A-C), and the cobalamin-independent methionine synthase (METE).b.Orthofinder2 14 produced gene of THI4 enzymes shows C. zofingiensis uniquely contains three copies of THI4 (labelled in red) c.Protein abundance of C. reinhardtii to −Fe for THIC, THI4, and METE in various organellar extracts (x-axis: chloroplast (CP), mitochondrion (MT) and whole cell (WC)) and +/-acetate 1 .d. Soluble proteome extract of C. reinhardtii in 0.25 µM Fe (limited) and 1 µM Fe (deficient) log2 ratio over 20 µM Fe (replete) 2 .METE and THI4 are highly depleted in 0.25 µM Fe.Cultures were grown in acetate and THIC abundance was not reported in publication.Source data are provided in the Source Data File.Supplementary Fig. 11 Transcriptome time courses of the de novo fatty acid synthesis pathway in C. zofingiensis and C. reinhardtii.